Third Generation Sequencing Technologies for Microbiome analysis
May 31 2018
Third generation technologyIllumina Synthetic Long read technology
SMRT-Single Molecule Long Read technology
Nanopore Technologies
Hi-C Technology
Comparison of all technologies
Metagenomic sequencing (including 16s and whole genome sequencing), metatranscriptomics are common techniques applied to study microbial communities and their role in maintaining host health or in ecological stability. Popular sequencing technologies in
Third Generation technologies
Third generation sequencing technologies have had a drastic impact on eukaryotic studies, in improving reference genomes providing assemblies with near chromosome level completion (link here). Here is another blog post we already have on third-generation sequencing technologies, explaining each technology, their comparison, costs and some research papers that have applied them.
Stemming from the above
Illumina Synthetic Long read technology
This method is more or less the same as the current HiSeq Illumina technology with a difference in the manner of barcoding to get longer reads, here is a brief description
- Cost: ~$2500 per GB
- Bioinformatics software: open source software are available
- Research papers: Here is a pdf provided by Illumina with
list of reviews and research studies using the long read technology
PacBio (SMRT-Single Molecule Long Read technology)
This method uses hairpin adaptors to attach the double strands and make circular DNA, and a polymerase sequence the entire circular strand over and over again, providing good coverage. Goto here for a brief description of how the technique works. They have three workflows, including full-length 16s sequencing, for metagenomics profiling and shotgun metagenome assemblies of near-complete genomes. Here is a link to PacBio’s workflows for microbial communities for all the three workflows. The kits offer up to ~10kbp read lengths, up to 100 fold coverage for 16s and ~50 per genome, for more information - click here or here.
- The estimated cost of the process: $300-$400 per GB (source)
- Bioinformatic software: Open source tools available: SMRT Tools or PacBio DevNet
- Research papers - Here is a link from the PacBio website
Nanopore Technologies
- Cost: $49.99 to $99.99 per Gb for MinIon - (source)
- Bioinformatics software: Available on
github :canu assembler,poretools , compiled list of tools. - Research papers: List from nanopore website
Hi-C Technology
Phase Genomics, have developed a ProxiMeta kit for metagenomic analysis. This method extracts the DNA sequence to keep the linkage between DNA molecules while sequencing to get
- Cost: Sequencing prep kit is 2000$ for 2 samples. The sequencing itself can be run on Illumina sequencers.
- Bioinformatics programs: ProxiMeta software (proprietary), there are open source programs available on
github and other tools with active development. - Research papers: cow gut microbiome study, human microbiome studies
10X Technology
Applies Illumina short read technology but keeping track of the reads can be assembled to a larger template. For more information about the method- go here. Like the Illumina long read technology, the sequencing prep kits are not distinct for microbial sequences specifically.
- Cost: -
- Bioinformatics: Supernova for assembly, LongRanger analysis pipelines, another cloud-based assembler developed for microbiome sample Athena
- Research papers: Human gut metagenome studies
Comparison of all technologies
See table